Method of applying a biological specimen to an analytic plate

ABSTRACT

A method applying a biological specimen to an analytic plate by using an applicator device to apply a coating to the analytic plate and adhering the biological sample to the plate. The coating is substantially transparent, translucent or invisible, and is substantially flush with the surface of the analytic plate. The coating is preferably comprised of a polysiloxane, siloxane, silicone, a silane, a silicon fluid, or a combination thereof and optionally an acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 12/624,202,filed Nov. 23, 2009, now U.S. Pat. No. 8,192,994, which is acontinuation-in-part of U.S. Ser. No. 10/989,785, filed Nov. 16, 2004,now abandoned, which is a continuation of U.S. Ser. No. 10/805,777,filed Mar. 22, 2004, now abandoned, which is a divisional application ofU.S. Ser. No. 10/225,412, filed Aug. 20, 2002, now U.S. Pat. No.6,713,304, which is a continuation-in-part of U.S. Ser. No. 09/373,468,filed Aug. 12, 1999, now U.S. Pat. No. 6,555,384, which is acontinuation-in-part of U.S. Ser. No. 09/021,077, filed Feb. 10, 1998,now U.S. Pat. No. 5,948,685, and which is also a continuation-in-part ofInternational Application No. PCT/US99/02854 filed Feb. 9, 1999. Each ofthe patents or applications listed above is hereby expresslyincorporated by reference herein in its entirety.

BACKGROUND

The present invention relates generally to the field of applyingbiological specimens analytic plates.

Standard microscope slides and diagnostic plates are thin, typicallyrectangular, sheets of glass, ceramic, or plastic. In use, a samplecomprising an aqueous or non-aqueous liquid liquid reagent, biologicalfluid and/or biological tissue section(s) is placed upon a portion ofthe slide or diagnostic glass plate. Before analysis, the sample on theslide or plate may be dried, placed in a fixative, or remain fresh priorto treatment for enhanced visualization by light, electron, orfluorescent microscopy, and/or including gross analysis with the humaneye. The sample may be analyzed in its natural state or may needtreatment with one or more liquid dyes to enhance visualization. Furthertreatment with molecular biological techniques may include, for example,treatment by monoclonal, polyclonal antibodies, in-situ hybridization bymolecular probes, and/or their liquid detection reagents. During routineanalysis or manipulation of a slide or plate, the sample or liquidreagent may spill from the slide, run or migrate onto other portions ofthe slide, and/or “wick off” if the slide touches another object, thusresulting in a loss of all or part of the liquid sample or reagent. Itis desirous to avoid such inadvertent or undesired mixing orcontamination of different samples or liquid reagents.

It is therefore beneficial for the slide or diagnostic plate to havemeans to confine the sample or liquid used in treating the sample to aspecific area on the slide or plate. This has been accomplishedpreviously by creating a slide or plate having one or more depressions,or “wells” therein. Alternatively, a physical barrier or hydrophobicmaterial may be applied to the slide surface in a bordered pattern toconfine the liquid applied to the plate within the area surrounded bythe border. Such borders may comprise a coating of Teflon, paint, wax,paraffin, epoxy resin, or other resinous material, or a paint. Each ofthese materials results in a border having a thickness resulting in araised border extending a distance above the surface of the glass, forexample, a Teflon layer is generally from about 0.001 to about 0.0025inches high. These raised areas are generally opaque and the end resultis a loss of the transparent nature of the slide where the border isapplied. In spite of the fact that these raised borders may be somewhateffective in confining the liquid, there continues to be a need for aslide or plate which achieves confinement of the liquid upon a slidewhile maintaining transparency of the glass or plate. It is the objectof the present invention to provide such a slide.

SUMMARY OF THE INVENTION

The present invention contemplates an analytic plate such as amicroscope slide or a diagnostic plate having a containment border forinhibiting migration of liquids or liquid samples thereon, wherein theborder is substantially transparent and is substantially flush with thesurface of the slide or plate and which covers only a portion of thesurface of the slide or plate, and a method of making such a plate orslide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a plan view of a microscope slide constructed in accordancewith the present invention.

FIG. 1B is a side view of the slide of FIG. 1A.

FIG. 2A is a plan view of another version of a microscope slideconstructed in accordance with the present invention.

FIG. 2B is a side view of the slide of FIG. 2A.

FIG. 3 is a plan view of another version of a microscope slideconstructed in accordance with the present invention.

FIG. 4 is a plan view of another version of a microscope slideconstructed in accordance with the present invention.

FIG. 5 is a plan view of another version of a microscope slideconstructed in accordance with the present invention.

FIG. 6 is a plan view of another version of a microscope slideconstructed in accordance with the present invention.

FIG. 7 is a perspective view of a pen used in accordance with thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention contemplates an analytic plate such as amicroscope slide or a diagnostic plate having a containment border forinhibiting migration of liquids or liquid samples thereon, wherein theborder is substantially transparent and is substantially flush with thesurface of the slide or plate and which covers only a portion of thesurface of the slide or plate.

Where used herein, the term “analytic plate” refers to those types ofplates such as microscope slides and diagnostic plates which are used,for example, in microscopic analysis or diagnostic analysis orcomparison of samples. Analytic plates are generally comprised of glassor plastic (translucent, transparent, or opaque) but may also compriseceramic materials. The analytic plates contemplated herein can have anyshape suitable for use in analytic devices wherein the analytic platecan hold or contain a liquid or sample for analysis. When used herein,the terms, “plate” and “slide” are intended to be interchangeable.

Referring now to FIGS. 1A and 1B, a glass microscope slide having thegeneral reference numeral 10 is shown. The slide 10 has a conventionallength, width and thickness as is well known to one of ordinary skill inthe art. The slide 10 has an upper surface 12 and a lower surface 14.Disposed upon a portion of the upper surface 12 is a liquid containmentborder 16 which in the version of FIG. 1A has a rectangular shape. Whereused herein the term “liquid containment border” or “containment border”refers to a transparent border which prevents passage of an aqueous ornon-aqueous liquid thereacross. The containment border 16 surrounds acontainment area 18 of the upper surface 12 of the slide 10. Thecontainment border 16 forms a liquid barrier about the containment area18. When a liquid or liquid sample (not shown) is placed upon thecontainment area 18 of the slide 10 for analysis, the containment border16 prevents the spreading, leakage or migration of the liquid or liquidsample from the containment area 18, thus causing the sample to beretained in a discrete and confined location upon the slide 10. Whereused herein, the term liquid or liquid sample is intended to refer to aliquid material, or a liquid biological sample (e.g., blood, urine,plasma, or cerebrospinal fluid) which is desired to be localized on theslide.

The coating material which is used to form the containment border 16comprises a material which when applied to the slide 10 is preferablytransparent or clear although it may have a color to indicate itsposition on the slide or have printed, by one of ordinary skill in theart, on the lower surface 14 and/or upper surface 12 of the slide 10information (lines or numbers or symbols) indicating the position of thecontainment border 16 on the upper surface 12. The containment border 16forms a molecular layer when dry and therefore is substantially flush(level) with the upper surface 12 of the slide 10. The containmentborder 16 is therefore not raised above the upper surface 12 to a degreethat is visible to the naked eye. In fact, the containment border 16preferably has a thickness of less than 0.0001 inch and more preferablyless than 0.00001 inch. After the coating material is applied to theslide 10 thereby forming the containment border 16, the slide 10 isdried. The containment border 16 may be transparent or translucent afterapplication, wherein it may be desired to buff or treat the slide 10chemically (e.g., by xylene, alcohol, or acetone, or other commonly usedlaboratory solvents) such that the containment border 16 is preferablyrendered clear (transparent) and invisible whereby the containmentborder 16 leaves the refractive index of the slide 10 unaltered whenviewed through a microscope.

In a preferred embodiment the coating material which forms thecontainment border 16 is a composition comprising a liquid repellantcompound dissolved in a volatile solvent. In a particularly preferredversion, the composition comprises an alkyl polysiloxane and a mineralacid mixed with a solvent in a manner well known in the art. Such amixture is described in U.S. Pat. No. 3,579,540, the specification ofwhich is hereby incorporated herein by reference in its entirety. Otherpolysiloxanes, siloxanes, silanes, silicones, silicon fluids, andcombinations thereof which can permanently or at least substantiallypermanently bond to a glass surface and function in accordance with thepresent invention are also contemplated and are well known in the art,and are available commercially for use herein. Although a polysiloxaneacid mixture is particularly preferred, it will be understood by one ofordinary skill in the art that any material which can adhere to thesurface of at least one of a glass, plastic or ceramic slide or plateand which forms a substantially non-raised molecular layer as describedand claimed herein and is suitable for use in the present invention.

It is generally necessary to apply a coating material compositioncomprising at least 1% by weight of the polysiloxane, siloxane, silane,silicone, silicon fluid or combinations thereof to provide a containmentborder 16 having sufficient liquid repellancy properties for the slide10 with the containment border 16 thereon to function as contemplated inthe present invention herein. More preferably the composition of thecoating material comprises at least 2.5%, 5%, 10%, 15%, 20% or 25% ofthe polysiloxane, the siloxane, the silane, the silicone, the siliconfluid, or combinations thereof.

The coating material can be applied to the slide 10 in any manner knownin the art for applying a liquid to a surface, for example, by brushing,wiping, by using a stamping device, by spraying or by application from adevice (such as a pen or pen-like device) filled with the coatingmaterial to be applied to the slides or plates (described in more detailbelow).

In an alternative method of application of the coating material for thecontainment border 16, the slide 10 may be provided with a removableraised layer of a material such as a silicone rubber which is applied asa raised strip on a portion of the upper surface 12 of the slide 10 (notshown). Prior to the application of the liquid or liquid sample fortreatment, the raised silicone strip is peeled away from the uppersurface 12 of the slide 10, leaving a residual coating comprising thecontainment border 16 in accordance with the present invention. Afterthe raised silicone rubber strip has been peeled away leaving thecontainment border 16, the slide 10 can be used in accordance with thepresent invention.

It is another distinctive characteristic of the present invention that,in a preferred embodiment, after the coating material is applied to theslide 10 to form the containment border 16 and the coating material hasdried thereon, the containment border 16 is highly resistant to abrasionand to chemical removal and physical removal by washing, scrubbing,wiping, placement in organic solvents, inorganic solvents, and aqueoussolvents. The slide 10 can therefore be used repeatedly without thecontainment border 16 losing its repellant properties.

The containment border 16 of the present slide 10 is furtherdistinguished from prior art slides which have Teflon borders or otherphysical barriers because the surface of such prior art slides must betreated before the Teflon coating can adhere to the slide (e.g., usingan adhesive) thus causing solvents to dissolve the adhesive and thesubsequent loss of the border's efficiency due to peeling and/or loss ofthe liquid confinement integrity of the border. The borders of slidesusing coatings of Teflon, epoxy, or paint are generally opaque and areraised above the surface of the slide, unlike the borders on the slidesof the present invention. In the present invention, there is nointervening layer (e.g., an adhesive) between the glass and the coatingcomprising the containment border 16. Further, borders of such prior artslides also suffer from non-specific binding of reagents along theirraised edges thereby causing interference with the specimen.

For example, interference may occur when the reagents used in processingbiological samples adhere, are drawn to, or react with these raisedborders causing undesirable background staining around the specimen.Additionally, these raised borders cause “pooling” of reagents in the“well” formed by the raised border, making rinsing of reagents moredifficult than the non-raised borders of the present invention.

The present invention also enables the user to coverslip the slides withknown manual and automated coversliping methods without the need to addexcess mountant into the space within the raised wells to fill the gapsof “dead air” space resulting from the raised borders. These “dead air”spaces, if not filled with mountant, would have bubbles present underthe coverslip, which is undesirable. The substantially non-raisedborders of the present invention do not interfere with the placement ofthe coverslip on the slide nor do they necessitate the addition ofmountant beyond the normal amount of mountant necessary for a normaluntreated slide to be coversliped.

Another advantage of the slide of the present invention relates to thetransparent nature of the border. Recent developments in image analysishas advanced specimen scanning to offer a reproducible analysis ofspecimens on a slide. These instruments automatically orsemi-automatically scan the slide to find the specimen and measurevariables. The need to maintain the transparent nature of the slide iscritical. The camera scans the slides and picks up anything that altersthe light path, therefore, it is important that the only object on theslide which alters the light passing through the camera is the specimenitself, not an opaque border that would typically block the entire lightpath.

Although the microscope slide of the present invention may consistsolely of a slide 10 with the containment border 16 thereon, in someembodiments the slide may further have a distinct marking surfacethereon for writing upon or for attaching a label thereto. FIGS. 2A and2B show such a slide, designated therein by the general referencenumeral 10 a. The slide 10 a has a marking surface 20 which is a“frosted” portion of the slide 10 a (i.e., a portion of the slide 10 awhich has been etched off or abraded). In an alternative version of sucha slide, the marking surface 20 may be an opaque epoxy or paintedcoating. Other means of forming a marking surface will be apparent toone of ordinary skill in the art. FIG. 2A further shows an alternativeversion of the invention wherein the containment border, designated bythe general reference numeral 22 comprises a pair of strips extendingfrom one edge of the slide to another, rather than forming a box patternas shown in slide 10 a.

FIG. 3 shows a slide 10 b which is essentially the same as slide 10 aexcept the containment border is a border 24 which forms an entire “box”on the surface 12 of the slide 10 b. FIG. 4 shows an alternativeembodiment of the invention, a slide 10 c having a containment border 26which comprises a pair of separate containment areas 28. The separatecontainment areas 28 can therefore contain separate samples which areprevented from mixing by the portion 30 of the containment border 26which separates the two containment areas 28. Although not specificallyshown in the figure, the slide 10 c may be constructed to comprise aplurality of separate containment areas 28 greater than two for holdinga plurality of samples, as will be understood by a person of ordinaryskill in the art.

FIG. 5 shows a slide 10 d having a pair of circular containment borders32 which surround containment areas 34. Alternative versions of slide 10d may have only a single circular containment border 32, or may have aplurality of circular containment borders 32. FIG. 6 shows a slide 10 ecomprising a containment border 36 having a diagonal border 38 extendingthereacross forming a pair of triangle shaped containment areas 40.Alternative versions of the slide 10 e may have only a single triangleshaped containment area 40, or may have a plurality of areas 40.Further, it will be understood by a person of ordinary skill in the artthat the shapes of the containment areas are not limited only to thoseshown in the figures herein. The containment areas may have othershapes, such as ovals, stars, ellipses, pentagons, hexagons, trapezoids,or even non-geometric or fanciful shapes. Further, a single slide mayhave more than one particular shape of containment border disposedthereon, for example, a circle and a box or a pair of circles and a pairof boxes.

As is evident from the above, each slide contemplated herein has only aportion of its surface having a coating made from the coating material,with the specific purpose for retaining a biological sample upon adiscrete and predetermined portion of the slide.

In an alternative embodiment of the invention, one or more of themicroscope slides or plates contemplated herein may be supplied as a kitalong with other components used in microscopic analysis of samples.Said other components may comprise stains and reagents commonly used bythose of ordinary skill, including but not limited to, stains, dyes,molecular biological reagents including monoclonal and polyclonalantibodies, and molecular probes and their detection reagents, and otheraqueous and non-aqueous processing reagents. Examples of aqueous andnon-aqueous processing reagents include xylene, toluene, acetone, andother organic and inorganic solvents, and alcohols, biological buffers,and aqueous reagents for use with antibodies, and molecular probes andtheir detection reagents.

As noted above, the containment border may be applied via a pen, orpen-like device, an example of which is shown in FIG. 7. The pen isdesignated by reference number 50 and comprises a body 52 having areservoir therein (not shown) which contains a quantity of the liquidcomposition comprising a coating material described elsewhere herein(e.g., polysiloxane). The pen 50 further comprises an applicator end 54,and a cap 56 for inhibiting evaporation of the coating material ordrying of the tip 54. The pen 50 or cap 56 may comprise means forclipping, e.g., to a pocket. The applicator end may be a brush, a swab,a rubber tip, or any other device known to one of ordinary skill in theart of applicator pens.

As contemplated herein, a user can use the pen 50 to custom make his own“bordered slides” having a containment border as described herein. Theborder applied in such a manner is resistant to removal by organicsolvents such as xylene, as described above. In use, the user applies alayer of the coating material, e.g., polysiloxane, to a slide, allows itto dry, then applies the aqueous or non-aqueous liquid or histologicalmaterial, or other biological sample, and carries out various processingsteps known in the art for analyzing the specimen (e.g., treating withstains and organic solvents). Alternatively, the sample may be appliedto the slide before the containment border is applied to the slide,e.g., a list of any tissue sample or any other biological sample thatrequires air drying before processing. Treatment with organic solventsused in the processing steps has substantially no effect on the durablecontainment border as claimed herein. The pen applicator of the presentinvention differs from other pen applicators known in the art (e.g., PAPPen) because such prior art pens are used only to apply a greasy or oilylayer to the slide which is neither resistant to abrasion or rubbing norresistant to organic solvents, i.e., the layer can be physically wipedor worn off and is not resistant to most organic solvents such asxylene. The containment borders provided by using the pen 50 describedherein are resistant to abrasion or to removal by organic solvents.Further, in an especially preferred version of the present invention,the coating material used in the pen 50 does not contain or comprise anoil.

The examples described herein are not intended to limit the scope of theinvention. Changes may be made in the construction and the operation ofthe various components, elements and assemblies described herein or inthe steps or the sequence of steps of the methods described hereinwithout departing from the scope of the invention as defined in thefollowing claims.

1. A method of applying a biological sample to an analytic plate,comprising: using an applicator to apply a fluid composition to asurface of a glass, plastic, or ceramic analytic plate, wherein theapplicator is a pen or pen-like device having a body having a reservoirtherein and has an applicator end with a tip constructed of a swab,brush, or rubber material, the reservoir containing a fluid composition,the fluid composition comprising a polysiloxane, a siloxane, a silane, asilicone, a silicon fluid, or a combination thereof, and wherein the tipof the applicator end extends from the body; and wherein the fluidcomposition is applied to the surface of the glass, plastic or ceramicanalytic plate by appressing the tip of the applicator end to thesurface of the glass, plastic or ceramic analytic plate wherein thefluid composition flows from the reservoir in the body through the tipand is disposed upon the surface of the glass, plastic or ceramicanalytic plate to form a coating thereon and wherein the coating has athickness of less than 0.0001 inch after drying; and applying abiological sample to the surface of the glass, plastic or ceramicanalytic plate after the coating has been applied to the glass, plasticor ceramic analytic plate, wherein the biological sample is caused to beadhered to the glass, plastic, or ceramic analytic plate.
 2. The methodof claim 1 comprising the additional step of wiping the fluidcomposition upon the surface of the glass, plastic or ceramic analyticplate after the fluid composition has been disposed upon the surface ofthe glass, plastic or ceramic analytic plate.
 3. The method of claim 1wherein the coating has a thickness of 0.00001 inch or less.
 4. Themethod of claim 1 wherein the fluid composition of the applicatorfurther comprises an acid.
 5. The method of claim 1 wherein the coatingis colorless.
 6. The method of claim 1 wherein the coating istransparent, translucent or invisible.
 7. The method of claim 1 whereinthe coating is transparent, translucent or invisible and is highlyresistant to removal or abrasion.
 8. The method of claim 1 wherein thecoating leaves the refractive index of the analytic plate unaltered whenviewed through a microscope when the analytic plate is glass or plastic.9. The method of claim 1 comprising the step of treating the analyticplate with an acid or base prior to the step of applying the coatingupon the surface of the analytic plate.
 10. The method of claim 1wherein the applicator is absent an oil in the reservoir.
 11. A methodof applying a biological sample to an analytic plate, comprising: usingan applicator to apply a fluid composition to a surface of a glass,plastic, or ceramic analytic plate, wherein the applicator is a pen orpen-like device having a body having a reservoir therein and has anapplicator end with a tip, the reservoir containing a fluid composition,the fluid composition comprising a polysiloxane, a siloxane, a silane, asilicone, a silicon fluid, or a combination thereof, and wherein the tipof the applicator end extends from the body; and wherein the fluidcomposition is applied to the surface of the glass, plastic or ceramicanalytic plate by appressing the tip of the applicator end to thesurface of the glass, plastic or ceramic analytic plate wherein thefluid composition flows from the reservoir in the body through the tipand is disposed upon the surface of the glass, plastic or ceramicanalytic plate to form a coating thereon and wherein the coating has athickness of less than 0.0001 inch after drying; and applying abiological sample to the surface of the glass, plastic or ceramicanalytic plate after the coating has been applied to the glass, plasticor ceramic analytic plate, wherein the biological sample is caused to beadhered to the glass, plastic, or ceramic analytic plate.
 12. The methodof claim 11 comprising the additional step of wiping the fluidcomposition upon the surface of the glass, plastic or ceramic analyticplate after the fluid composition has been disposed upon the surface ofthe glass, plastic or ceramic analytic plate.
 13. The method of claim 11wherein the coating has a thickness of 0.00001 inch or less.
 14. Themethod of claim 11 wherein the fluid composition of the applicatorfurther comprises an acid.
 15. The method of claim 11 wherein thecoating is colorless.
 16. The method of claim 11 wherein the coating istransparent, translucent or invisible.
 17. The method of claim 11wherein the coating is transparent, translucent or invisible and ishighly resistant to removal or abrasion.
 18. The method of claim 11wherein the coating leaves the refractive index of the analytic plateunaltered when viewed through a microscope when the analytic plate isglass or plastic.
 19. The method of claim 11 comprising the step oftreating the analytic plate with an acid or base prior to the step ofapplying the coating upon the surface of the analytic plate.
 20. Themethod of claim 11 wherein the applicator is absent an oil in thereservoir.